Octanal

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

publication dated 1994

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Cytotoxic and genotoxic effects of five n-alkanals in primary cultures of rat and human hepatocytes

Author:

Martelli A, Canonero R, Cavanna M, Ceradelli M & Marinari UM

Year:

1994

Bibliographic source:

Mutation Research 323 (1994) 121-126

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Nonanal

EC Number:

204-688-5

EC Name:

Nonanal

Cas Number:

124-19-6

Molecular formula:

C9H18O

IUPAC Name:

nonanal

Details on test material:

One of five n-alkanals - propanal, butanal, pentanal, hexanal and nonanal.

More than 300 aldehydes have been identified in foods and some chemicals of this family have been found to constitute the terminal non-radical products of the free radical induced peroxidative breakdown of biomembrane polyunsaturated fatty acids. Due to the electrophilic nature of the carbonyl carbon, aldehydes have been shown to react with thiols and amines and to form protein-protein, DNA-protein and DNA-DNA cross-links.

Propanal (98% pure), butanal (99% pure), pentanal (98% pure), hexanal (98% pure), nonanal (98% pure) and N-nitrosodimethylamine (NOMA) were purchased from E. Merck, Darmstadt, Germany.

Other materials used in the assay were collagenase type IV from Sigma Chimica, Italy; Williams' E medium from Flow Laboratories, Milan, Italy and [methyl-3H] thymidine (specific activity 23-25 Ci/mmole) from Amersham International (UK). All other chemicals, reagent grade, were obtained from E. Merck.

Test animals

Species:

other: rat and human hepatocytes

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

The liver has the most comprehensive metabolic activity, the highest exposure to aldehydes ingested with foods, and the greatest susceptibility to lipid peroxidation, it was considered of interest to examine the five above mentioned n-alkanals in primary cultures of hepatocytes for both cytotoxicity and capability of inducing unscheduled DNA synthesis (UDS). Due to the known species differences in the activity of aldehyde dehydrogenases responsible for the oxidation to carboxylic acids of the functional group of these compounds a comparison has been carried out between rat and human hepatocytes.

Rat hepatocytes were isolated from Sprague-Dawley male albino rats (200-250 g) by collagenase perfusion. Cell suspensions less than 80% viable, as evaluated by the trypan blue exclusion test, were discarded.
Human hepatocyte suspensions were prepared from apparently healthy fragments of human liver discarded during the course of prescribed surgery from two human subjects. As checked by both macroscopic and histological examinations, these fragments were devoid of appreciable alterations. The proportion of viable hepatocytes after perfusion was 65% in case 1 and 83% in case 2.

Administration / exposure

Route of administration:

other: cell culture exposure

Vehicle:

Isolated rat or human hepatocytes were suspended in William's E supplemented with 10% calf serum and gentamicin (50µg/mI), and plated at a concentration of 1 x 10E6 in 35-mm Petri dishes coated with rat tail collagen. After 3 h attachment, dishes were washed with WE and refed with serum-free WE supplemented with half-log-spaced concentrations of the n-alkanals.

NOMA was used at a dose of 5 mM as the positive control to verify the metabolic competence of hepatocytes. At the end of a 20-h exposure
the cytotoxic effect of each concentration tested was immediately evaluated by washing the dishes with saline (0.85% NaCO and adding 0.8 mI/dish of a 0.4% trypan blue solution in saline; the percentage of viable cells was calculated by counting in duplicate 1000 cells/dish.

Details on exposure:

Cultures were exposed simultaneously for 20 h to n-alkanals and 10µCi/ml [methyl³H] thymidine and were processed immediately after treatment for the autoradiographic evaluation of UDS. Each dose of n-alkanals was evaluated in two independent experiments carried out using cultures prepared from two rat or two human donors. Data are expressed as the mean ± SD of the 200 net nuclear counts obtained from four autoradiographs
(two from each donor). Silver grains over the nucleus minus the grains over a randomly chosen equal-sized area in the cytoplasm were defined as net grains per nucleus. Cytoplasmic labeling was also considered in order to assess a possible effect of the five n-alkanals on mitochondrial
DNA.

Duration of treatment / exposure:

20 hour culture exposure

Frequency of treatment:

Single treatment, experiments were replicated

Post exposure period:

No data

No. of animals per sex per dose:

Not applicable - in vitro cultures used

Positive control(s):

No data

Examinations

Tissues and cell types examined:

Cultures were exposed simultaneously for 20 h to n-alkanals and 10µCi/ml [methyl³H] thymidine and were processed immediately after treatment for the autoradiographic evaluation of UDS. Each dose of n-alkanals was evaluated in two independent experiments carried out using cultures prepared from two rat or two human donors. Data are expressed as the mean ± SD of the 200 net nuclear counts obtained from four autoradiographs
(two from each donor). Silver grains over the nucleus minus the grains over a randomly chosen equal-sized area in the cytoplasm were defined as net grains per nucleus. Cytoplasmic labeling was also considered in order to assess a possible effect of the five n-alkanals on mitochondrial
DNA.

Details of tissue and slide preparation:

Cultures were exposed simultaneously for 20 h to n-alkanals and 10µCi/ml [methyl³H] thymidine and were processed immediately after treatment for the autoradiographic evaluation of UDS. Each dose of n-alkanals was evaluated in two independent experiments carried out using cultures prepared from two rat or two human donors. Data are expressed as the mean ± SD of the 200 net nuclear counts obtained from four autoradiographs
(two from each donor). Silver grains over the nucleus minus the grains over a randomly chosen equal-sized area in the cytoplasm were defined as net grains per nucleus. Cytoplasmic labeling was also considered in order to assess a possible effect of the five n-alkanals on mitochondrial
DNA.

Evaluation criteria:

No information

Statistics:

No information

Results and discussion

Test results

Sex:

not specified

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After 20 h exposure, cytotoxicity was similar in cells of the two species, and increased with the length of the carbon chain. In rat hepatocytes, propanal (10-100 mM), butanal (10 -100 mM), pentanal (0 -30 mM) and hexanal (3-30 mM) induced a modest but significant and dose-dependent increase of net nuclear grain counts, while in human hepatocytes this effect was not detected. Nonanal (3-30 mM), which showed the highest cytotoxic effect, failed to induce UDS in both cell types. These results seem to suggest that at the concentrations which are presumably attained after ingestion with food or generated by lipid peroxidation processes the five n-alkanals tested are presumably unable to induce genotoxic effects in the human liver.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The alkanals propanal, butanal, pentanal, hexanal and nonanal: (a) produce a cytotoxic effect in primary cultures of both rat and human
hepatocytes which is similar in both species and which increases with the length of the carbon chain (b) induce, with the exception of nonanal, a modest but significant and dose-dependent amount of DNA repair synthesis in rat, but not in human, hepatocytes.

In conclusion, the following considerations suggest that the probability of occurrence in humans of genotoxic effects produced by the five n-alkanals considered in this study is negligible. In the first place, even if they were weak inducers of mutations and DNA repair in rodent cells, their active concentrations were far higher than those which can be attained in vivo as a consequence of ingestion with food or generation by lipid peroxidation processes. In the second place, human cells seem to be less sensitive to n-alkanal genotoxicity than rodent cells under normal conditions of efficiency of detoxification systems

Executive summary:

Five n-alkanals were examined for cytotoxicity, as evaluated by the trypan blue exclusion test, and for genotoxicity, as evaluated by the induction of unscheduled DNA synthesis (UDS), in primary cultures of rat and human hepatocytes. After a 20 hour exposure, cytotoxicity was similar in cells of the two species, and increased with the length of the carbon chain. In rat hepatocytes, propanal (10-100 mM), butanal (10 -100 mM), pentanal (0 -30 mM) and hexanal (3-30 mM) induced a modest but significant and dose-dependent increase of net nuclear grain counts, while in human hepatocytes this effect was not detected. Nonanal (3-30 mM), which showed the highest cytotoxic effect, failed to induce UDS in both cell types. These results seem to suggest that at the concentrations which are presumably attained after ingestion with food or generated by lipid peroxidation processes the five n-alkanals tested are presumably unable to induce genotoxic effects in the human liver.